Use of Mild Electrophiles to Reduce Artifacts in Analyzing Glycans Released from Glycoproteins or Glycopeptides

ABSTRACT

The presence of mild electrophiles, such as aldehydes, during the denaturation of glycoproteins or glycopeptides and subsequent enzymatic deglycosylation reduces artifacts in subsequent analyses of the glycans released from the glycoproteins or gly-copeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/376,344, filed Aug. 17, 2016, the contents of whichare incorporated herein by reference for all purposes.

STATEMENT OF FEDERAL FUNDING

Not applicable.

BACKGROUND OF THE INVENTION

This invention relates to the field of improving labeling ofnucleophilic biomolecules by electrophilic labels, and particularly tolabeling glycosylamines.

Many of the proteins produced by eukaryotic cells are modified aftertranslation by the addition of covalently-linked, linear or branchedchains of carbohydrates. These protein-carbohydrate conjugates arereferred to as glycoproteins; the point at which the carbohydrate isattached is referred to as a glycosylation site. Attachedpolysaccharides or oligosaccharides are referred to as glycans. A widerange of glycans are found on the different glycosylation sites ofparticular glycoproteins. The particular pattern of glycans on aparticular glycoprotein is determined by the specific cell line thatproduced the protein and the conditions under which the cells weregrown.

Since the glycans conjugated to a protein can affect characteristicscritical to its function, including pharmacokinetics, stability,bioactivity, or immunogenicity, it is important in many uses todetermine which glycans are present. Thus, the ability to remove some orall of the glycans from a protein and to analyze the glycans todetermine their composition is useful for determining whether theglycoprotein will have its desired effect. For example, the Food andDrug Administration (“FDA”) requires characterization of carbohydratesattached to biologics (such as therapeutic glycoproteins and vaccines)to show composition of matter and consistency of manufacture, resultingin a need for extensive characterization of the product. Analysis of theprofile of the released carbohydrates is also important for qualitycontrol in the production of recombinant proteins, in which a change incarbohydrate profile may indicate stress in the system, signalingconditions that may require a commercial-scale fermenter of expensiveprotein to be discarded. There is therefore considerable interest bybiochemists, clinical chemists and pharmaceutical manufacturers indetermining the distribution profiles of glycans in biological samples,such as therapeutic glycoproteins.

Glycans are typically attached to glycoproteins in one of two ways. Inthe first, referred to as N-glycans, the glycans are attached through anN-glycosidic bond at an asparagine residue. In the second, referred toas O-glycans, glycans are attached to an oxygen atom on an amino acidresidue. For example, N-acetyl-galactosamine can be enzymaticallyattached to an oxygen on a serine or a threonine residue.

N-glycans can be enzymatically released from glycoproteins by enzymaticcleavage by various enzymes, such as PNGase F(Peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase, EC 3.5.1.52.).Enzymatic digestion of N-glycans, such as by PNGase F, typically occursin an aqueous solution, and results in the initial release of theN-glycans as β-glycosylamines, in which the free-reducing end of thereleased glycan is conjugated with ammonia (see, e.g., Tarentino, et al.TIGG 1993, 23, 163-170; Rasmussen J. R. J. Am. Chem. Soc. 1992, 114,1124-1126; Risley, et al. J. Biol. Chem. 1985, 260, 15488-15494, 1985).PNGase F-released N-glycans can be labeled by reductive amination, inwhich the free-reducing end of a glycan is conjugated to the free aminogroup of a label, such as a fluorescent dye or a electrical charge, orby use of an amine-reactive dye. Depending on the label used, thelabeled glycans can then be analyzed by any of a variety of analyticalmethods, such as high performance liquid chromatography (“HPLC”),capillary electrophoresis (“CE”), carbohydrate gel electrophoresis, ormass spectrometry (“MS”). Labeling of N-glycans is taught, for example,in co-owned U.S. Pat. Nos. 8,124,792 and 8,445,292.

Many protocols use a so-called “one pot” procedure, in which glycans arereleased as glycosylamines, which are then labeled for analysis in thepresence of the now aglycosylated or partially glycosylatedglycoprotein. Unfortunately, proteins contain reactive nucleophilicN-termini and amino acids, such as lysines, which have nucleophilic sidechains. Since the aglycosylated or partially glycosylated glycoproteinis still present in the solution, the N-termini and reactive side chainsof the amino acids on the protein may also be labeled, resulting in aprotein with multiple labels. These labeled proteins may exhibit similarphysicochemical properties to the labeled glycosylamines. The labeledproteins may be difficult to separate from the labeled glycans, and maycoelute and comigrate on HPLC or capillary electrophoresis,respectively. This can result in artifacts that make it difficult toquantify the glycans of interest

There remains a need for compositions and methods that improve labelingof glycosylamines and other nucleophiles. Surprisingly, the presentinvention meets these and other needs.

BRIEF SUMMARY OF THE INVENTION

In a first group of embodiments, the invention provides in vitro methodsof labeling glycosylamines released from a glycoprotein or glycopeptideof interest, the method comprising: (a) forming a mixture of theglycoprotein or glycopeptide and a mild electrophile in a solution, (b)incubating the mixture in the solution at a first temperature and for atime sufficient to denature the glycoprotein or glycopeptide, (c)reducing the temperature of the mixture in the solution to a secondtemperature, which second temperature is suitable for enzymaticdeglycosylation of the glycoprotein or glycopeptide, (d) contacting theglycoprotein or glycopeptide at the second temperature with at least onedeglycosylation enzyme which releases glycans from a glycoprotein orglycopeptide as glycosylamines, thereby releasing glycans from theglycoprotein or glycopeptide as glycosylamines, and (e) contacting thereleased glycosylamines with an amine-reactive dye under conditionsallowing the amine-reactive dye to label the released glycosylamines,thereby labeling the released glycosylamines. In some embodiments, thefirst temperature is 90° C. or higher. In some embodiments, the solutionfurther comprises a detergent. In some embodiments, the at least onedeglycosylation enzyme which releases glycans from said glycoprotein orglycopeptide as glycosylamines is PNGase F. In some embodiments, themild electrophile is DL-glyceraldehyde, glyceraldehyde dimer,glycoaldehyde dimer, erythrose, pyruvaldehyde, 2-ethylbutyraldehyde,2-methylbutyraldehyde, 2-methylvaleraldehyde,4-methyl-4-nitrovaleraldehyde, acetaldehyde, butyraldehyde,isobutyraldehyde, propionaldehyde, or valeraldehyde. In someembodiments, the mild electrophile is DL-glyceraldehyde, glyceraldehydedimer, or glycoaldehyde dimer. In some embodiments, the mildelectrophile is DL-glyceraldehyde. In some embodiments, the mildelectrophile is glyceraldehyde dimer. In some embodiments, the mildelectrophile is glycoaldehyde dimer. In some embodiments, theglycoprotein of interest is an antibody. In some embodiments, thecontacting of released glycosylamines with the amine-reactive dye instep (e) is within 30 minutes of release from the glycoprotein orglycopeptide. In some embodiments, the contacting of releasedglycosylamines with the amine-reactive dye in step (e) is within 15minutes of release from the glycoprotein or glycopeptide. In someembodiments, the contacting of released glycosylamines with theamine-reactive dye in step (e) is within 1 minute of release of theglycosylamines from the glycoprotein or glycopeptide. In someembodiments, the amine-reactive dye is selected from the groupconsisting of (2-(diethylamino)ethyl4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate) (InstantPC™)8-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,5-trisulfonicacid (“Amine-reactive ANTS™”),4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoic acid(“InstantAA™”),(7-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,6-trisulfonicacid) (“InstantQ™”), and RAPIFLUOR-MS®. In some embodiments, thereleased glycosylamines are at or at about room temperature whencontacted with the amine-reactive dye. In some embodiments, the methodfurther comprises either: step (f), providing the labeled glycosylaminesto an analytical means, or, step (f′) subjecting the labeledglycosylamines to a solid phase extraction means that reduces the amountof mild electrophile present, to obtain labeled glycosylamines with areduced amount of the mild electrophile present, and step (f″) providingthe labeled glycosylamines with the reduced amount of the mildelectrophile present to an analytical means. In some embodiments, thesolid phase extraction means is a solid phase cartridge. In someembodiments, the analytical means is selected from the group consistingof high-pressure liquid chromatography, capillary electrophoresis,fluorescence analysis, mass spectrometry, and a combination of any ofthese. In some embodiments, the glycoprotein of interest is in abiological sample. In some embodiments, the biological sample isselected from the group consisting of: a cell culture supernatant, amembrane protein preparation, milk, a tissue sample, cerebrospinalfluid, plasma, serum, urine, a respiratory secretion, bile synovialfluid, pleural fluid, lymph, tears, saliva, and a stool sample. In someembodiments, the biological sample is milk.

In a further group of embodiments, the invention provides compositionscomprising: (i) a detergent, (ii) a mild electrophile, (iii) at leastone deglycosylation enzyme which releases glycans from the glycoproteinas glycosylamines, (iv) a partially or fully deglycosylated protein orpeptide from which glycans have been removed by the deglycosylationenzyme, and (v) glycosylamines released from the partially or fullydeglycosylated protein by the enzyme. In some embodiments, theglycoprotein is an antibody. In some embodiments, the deglycosylationenzyme is PNG F. In some embodiments, the mild electrophile isDL-glyceraldehyde, glyceraldehyde dimer, glycoaldehyde dimer, erythrose,pyruvaldehyde, 2-ethylbutyraldehyde, 2-methylbutyraldehyde,2-methylvaleraldehyde, 4-methyl-4-nitrovaleraldehyde, acetaldehyde,butyraldehyde, isobutyraldehyde, propionaldehyde, or valeraldehyde. Insome embodiments, the mild electrophile is DL-glyceraldehyde,glyceraldehyde dimer, or glycoaldehyde dimer. In some embodiments, themild electrophile is DL-glyceraldehyde.

In another group of embodiments, the invention provides in vitro methodsof reducing artifacts in analyzing glycans released from a glycoproteinor glycopeptide and labeled by reductive amination, said methodcomprising the following steps in the following order: (a) forming asolution comprising the glycoprotein or glycopeptide, and a mildelectrophile, under conditions allowing said mild electrophile to blockany nucleophilic components present in the solution or on theglycoprotein or glycopeptide,(b) heating the solution to a firsttemperature, and for a time, sufficient to denature the glycoprotein orglycopeptide, (c) reducing the temperature of the solution to a second,cooler temperature, (d) releasing glycans from said glycoprotein orglycopeptide, (e) labeling the glycans released from the glycoprotein orglycopeptide by reductive amination, and (f) analyzing the glycanslabeled by the reductive amination, whereby blocking of the nucleophiliccomponents present in the solution or on the glycoprotein orglycopeptide prior to the denaturation and reductive amination reducesartifacts in the analysis of the released glycans. In some embodiments,the mild electrophile is DL-glyceraldehyde, glyceraldehyde dimer,glycoaldehyde dimer, erythrose, pyruvaldehyde, 2-ethylbutyraldehyde,2-methylbutyraldehyde, 2-methylvaleraldehyde,4-methyl-4-nitrovaleraldehyde, acetaldehyde, butyraldehyde,isobutyraldehyde, propionaldehyde, or valeraldehyde. In someembodiments, the mild electrophile is DL-glyceraldehyde, glyceraldehydedimer, or glycoaldehyde dimer. In some embodiments, the mildelectrophile is DL-glyceraldehyde. In some embodiments, the mildelectrophile is glyceraldehyde dimer. In some embodiments, the mildelectrophile is glycoaldehyde dimer. In some embodiments, theglycoprotein is an antibody. In some embodiments, the glycoprotein ofinterest is in a biological sample. In some embodiments, the biologicalsample is selected from the group consisting of: a cell culturesupernatant, a membrane protein preparation, milk, a tissue sample,cerebrospinal fluid, plasma, serum, urine, a respiratory secretion, bilesynovial fluid, pleural fluid, lymph, tears, saliva, and a stool sample.In some embodiments, the biological sample is milk. In some embodiments,the solution further comprises a detergent. In some embodiments, theanalysis is selected from the group consisting of high-pressure liquidchromatography, capillary electrophoresis, fluorescence analysis, massspectrometry, and a combination of any of these.

In yet a further group of embodiments, the invention provides methods ofreducing artifacts in analyzing glycans released from a glycoprotein orglycopeptide without labeling, comprising the following steps in thefollowing order: (a) forming a solution comprising the glycoprotein orglycopeptide, and a mild electrophile, under conditions allowing themild electrophile to block any nucleophilic components present in thesolution or on the glycoprotein or glycopeptide, (b) heating thesolution to a first temperature, and for a time, sufficient to denaturesaid glycoprotein or glycopeptide, (c) reducing the temperature of saidsolution to a second, cooler temperature, (d) releasing glycans from theglycoprotein or glycopeptide, and (e) analyzing the released glycans,whereby said blocking of said nucleophilic components present in saidsolution or on said glycoprotein or glycopeptide prior to saiddenaturation reduces artifacts in said analysis of said releasedglycans. In some embodiments, the analysis is by mass spectrometry,High-performance Anion Exchange Chromatography coupled with PulsedAmperometric Detection (“HPAE-PAD”), Charged Aerosol Detection (“CAD”),Evaporative Light Scattering Detection (“ELSD”), or a combination of anyof these.

In still another group of embodiments, the invention provides kits forlabeling glycosylamines released from a glycoprotein or glycopeptide,comprising, (a) a mild electrophile, (b) an amine-reactive dye, an agentfor performing reductive amination, or both an amine-reactive dye and anagent for performing reductive amination, and (c) instructions ondenaturing said glycoprotein or glycopeptide in the presence of the mildelectrophile. In some embodiments, the agent for performing reductiveamination is anthranilic acid (“2-AA”). In some embodiments, the agentfor performing reductive amination is 2-aminobenzamide (“2-AB”). In someembodiments, the kit further comprises a deglycosylation enzyme. In someembodiments, the deglycosylation enzyme is PNGase F. In someembodiments, the mild electrophile is DL-glyceraldehyde, glyceraldehydedimer, glycoaldehyde dimer, erythrose, pyruvaldehyde,2-ethylbutyraldehyde, 2-methylbutyraldehyde, 2-methylvaleraldehyde,4-methyl-4-nitrovaleraldehyde, acetaldehyde, butyraldehyde,isobutyraldehyde, propionaldehyde, or valeraldehyde. In someembodiments, the mild electrophile is DL-glyceraldehyde, glyceraldehydedimer, or glycoaldehyde dimer. In some embodiments, the said mildelectrophile is DL-glyceraldehyde. In some embodiments, theamine-reactive dye is selected from the group consisting of(2-(diethylamino)ethyl4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate) (InstantPC™),8-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,5-trisulfonicacid (“InstantANTS™”),4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoic acid(“InstantAA™”),(7-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,6-trisulfonicacid) (“7-ANTS”), and RAPIFLUOR-MS®.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 is a graph of a HPLC analysis of molecules in a solutionresulting from the denaturing and deglycosylation of an exemplarglycoprotein, RNase b, in the absence (solid line) or presence (dashedline) of an exemplar mild electrophile, DL-glyceraldehyde (“Blocker”) inwater. Molecules present in the solution, including glycans releasedfrom the glycoprotein and any other molecules present in the solutionreactive with the label at the time of labeling were then labeled withthe label InstantPC™. The resulting solution was then subjected to aclean-up step to remove unbound label and larger proteins prior to HPLCanalysis. Y-axis: emission units (“EU”). X-axis: time, in minutes.

FIG. 2. FIG. 2 is a graph of a HPLC analysis of a solution resultingfrom the denaturing and deglycosylation of an exemplar glycoprotein,Bovine Thyrotropic Hormone, in the absence (solid line) or presence(dashed line) of an exemplar mild electrophile, DL-glyceraldehyde(“Blocker”) in water. Glycans released from the glycoprotein and anyother molecules present in the solution reactive with the label at thetime of labeling were then labeled with the label InstantPC™. Y-axis:emission units (“EU”). X-axis: time, in minutes.

FIG. 3. FIG. 3 is a graph of a capillary electrophoresis analysis withthe Y-axis of the two lines on the graph offset from one another so thatthe two lines can be more clearly seen. An exemplar glycoprotein, RNaseb, was denatured and deglycosylated in the absence (solid line) orpresence (dashed line) of an exemplar mild electrophile,DL-glyceraldehyde (“Blocker”) in water, and glycans released from theglycoprotein and any other molecules present in the solution reactivewith the label at the time of labeling were then labeled with the labelInstantQ™. Y-axis: relative fluorescence units (“RFU”). X-axis: Relativemigration time “RMT”.

FIG. 4. FIG. 4 is a graph of a HPLC analysis of the solution resultingfrom the denaturing and deglycosylation of an exemplar glycoprotein,epoetin alfa, in the absence (solid line) or presence (dashed line) ofan exemplar mild electrophile (“blocker”). Glycans released from theglycoprotein and any other molecules present in the solution reactivewith the label at the time of labeling were then labeled with the labelRAPIFLUOR-MS®. Y-axis: emission units (“EU”). X-axis: time, in minutes.The arrows denote areas in which artifacts in the HPLC solution havebeen diminished by the presence of the mild electrophile during thedenaturation and labeling steps compared to the solution in a mildelectrophile was not present.

DETAILED DESCRIPTION

Analysis of the glycans attached to glycoproteins or glycopeptides hasbecome important for meeting FDA requirements regarding showingcomposition of matter and consistency of manufacture of therapeuticglycoproteins and for providing quality control during the production ofrecombinant glycoproteins. As described in the Background, for speedconvenience and cost-savings, N-glycans present on a glycoprotein orglycopeptide of interest are often analyzed in “one pot” procedures byreleasing them from the glycoprotein by enzymatic digestion and thenlabeling the resulting released glycans, which are released asglycosylamines, with an amine reactive dye in the presence of thenow-aglycosylated or partially deglycosylated glycoprotein orglycopeptide. Unfortunately, glycoproteins and glycopeptides containnucleophilic N-termini and amino acids, such as lysines, withnucleophilic side chains, each of which can also react withamine-reactive dyes and be labeled by the dye. This can be a particularproblem with some negatively charged dyes used in capillaryelectrophoresis, or “CE” analysis. The labeled deglycosylated proteinsor peptides may be difficult to separate from the labeled glycans, andmay co-elute and co-migrate on HPLC or capillary electrophoresis,respectively and is particularly true for smaller, labelledaglycosylated proteins or peptides, as the dye changes the protein's orpeptide's migration characteristics. The labeling of the deglycosylatedprotein or peptide can result in artifacts that make it difficult toquantify the glycans of interest.

Surprisingly, it has now been found that mild electrophiles, such assmall, polar aldehydes, can be used to block the N-termini ofglycoproteins or glycopeptides, as well as nucleophilic side chains onamino acids such as lysines, and are particularly useful in blocking theN-termini and nucleophilic side chains or other nucleophilic componentsof glycoproteins or glycopeptides during heat denaturation.Glycoproteins or glycopeptides with the N-termini and nucleophilic sidechains blocked can then be subjected to deglycosylation and labeling ofthe released glycosylamines with amine-reactive dyes without having theamine-reactive dye also react with the protein's or peptide's blockedN-terminus and nucleophilic side chains. In studies underlying thepresent disclosure, a surprising and dramatic reduction in artifacts wasseen when analyzing the labeled glycans between samples denatured andthen deglycosylated and labeled in the presence of an exemplar mildelectrophile as compared to a like sample subject to the same protocol,but without the presence of the mild electrophile.

Embodiments of the inventive methods thus reduce the steps needed toanalyze glycans released from a glycoprotein or glycopeptide, while atthe same time improving the accuracy of the analysis. They reduce thenumber of steps needed to analyze released glycans by eliminating theneed for a step to remove aglycosylated or partially deglycosylatedprotein or peptide from the solution containing the released glycansprior to labeling with an amine-reactive dye to avoid having lysineresidues or other amino acids with a nucleophilic side chain (as well asthe free amino end of the protein or peptide) labeled by the dye. Theyimprove the accuracy of analysis of the glycans released from theglycoprotein or glycopeptide in two ways. First, they dramaticallyreduce artifacts from the labeling of lysine residues or other aminoacids with a nucleophilic side chain, or other nucleophilic componentsof the glycoprotein or glycopeptide, as well as the free amino end ofthe protein or peptide. Some analytical systems show such labeledproteins and peptides as “humps,” rather than sharp peaks, making itharder to quantitate labeled glycans that might co-migrate with thelabeled, deglcosylated protein or peptide. Decreasing the labeling ofthe protein or peptide by blocking the reactive amines with a mildelectrophile prior to the labeling reaction reduces or eliminates thisproblem. Second, when the mixture containing the blocked protein orpeptide and the mild electrophile is denatured prior to labeling of theprotein or peptide of interest, any remaining mild electrophile isavailable to react with any trace nucleophiles that may be present inthe denaturation mix, rendering the nucleophiles unavailable forreaction with the dye when the labeling step is commenced. This furtherreduces artifacts during the labeling step.

In some embodiments, the inventive methods comprise the following steps,in the following order. First, the mild electrophile and theglycoprotein or glycopeptide of interest are combined in a solution.Conveniently, this is done by adding the mild electrophile to a solutionalready containing the glycoprotein or glycopeptide of interest, but theglycoprotein or glycopeptide of interest may instead be added to asolution already containing the mild electrophile, or both may be addedto a suitable buffer. The resulting mixture, which may further includedetergents and other reagents conventionally used in denaturing proteinsor peptides in enzymatic deglycosylation protocols, is then heated todenature the glycoprotein or glycopeptide of interest. Without wishingto be bound by theory, it is believed that heat treatment makes thelysines and possibly the N-terminus of the glycoprotein or glycopeptidemore reactive, allowing them to form Schiff bases/ hemiaminals with thealdehydes. Without wishing to be bound by theory, it is further believedthat the mild electrophile attaches to lysine residues, othernucleophilic components present on the glycoprotein or glycopeptide, theprotein's or peptide's N-terminus, or combinations of any of these,blocking the lysine residues, other nucleophilic components, proteinN-terminus, or combination of any of these, during subsequent steps ofthe protocol under the reaction conditions to be employed. (Persons ofskill will appreciate that, while each glycoprotein or glycopeptidemolecule has only one N-terminus, a plurality of such molecules will bepresent in any deglycosylation protocol. Thus, a reference to blockingof a glycoprotein's or a glycopeptide's N-termini will be understood bythe reader to refer to the N-termini of a plurality of separateglycoprotein or glycopeptide molecules.)

The now-denatured glycoprotein or glycopeptide of interest is thencooled to a temperature suitable for enzymatic deglycosylation and thenput in contact with a deglycosylation enzyme selected by thepractitioner. For example, the deglycosylation enzyme may be added tothe mixture containing the now-denatured protein or peptide of interest,the mixture containing the now-denatured protein or peptide of interestmay be added to a reaction vessel containing the enzyme, or the mixturemay be flowed over a substrate on which the deglycosylation enzyme hasbeen immobilized. In some embodiments, the contacting of the mixturewith the deglycosylation enzyme takes place in a microfluidic apparatus.In some preferred embodiments, the enzyme is the amidase PNGase F, whichreleases N-glycans as nascent glycosylamines.

The glycosylamines released from the now fully- orpartially-aglycosylated protein or peptide of interest are then treatedwith an amine-reactive dye to label the glycosylamines for analysis.(For convenience of reference, both glycosylamines labeled byamine-reactive dyes and glycans labeled by reductive amination aresometimes referred to herein as “labeled glycans.” It will be clear incontext if a particular reference refers to only a labeled glycosylamineor a glycan labeled by reductive amination. It is further noted thatamine-reactive dyes have been used to label glycosylamines for years andconditions suitable for them to react with and to label glycosylaminesare well known in the art.) As the protein or peptide's lysine residuesand N-termini have been blocked by the mild electrophile, they are notlabeled with the amine-reactive dye. The blocked, aglycosylated proteinor peptide therefore does not have to be removed from the solution priorto labeling of the released, labeled glycans, as it will not be labeledand will not introduce artifacts which could otherwise add to thedifficulty of identifying or quantifying the labeled glycans.

In some embodiments, the mild electrophile is removed from the solutionbefore labeling the glycosylamines released from the glycoprotein orglycopeptide, such as with an amine-reactive dye. In some embodiments,it is not removed before labeling the glycosylamines. Without wishing tobe bound by theory, it is believed that, at the temperatures used inprotocols for labeling glycans (typically around 50° C. or lower), theamine-reactive dye outcompetes the mild electrophile in reacting withthe glycosylamines.

In some embodiments, the protocol described above may be used forlabeling glycans of glycoproteins in a biological sample, rather thanglycoproteins that have been purified to be free of the presence ofother types of biological molecules. Biological samples containingglycoproteins may come from, for example: cell culture supernatants;membrane protein preparations; milk (which contains glycoproteins andfree oligosaccharides); tissue samples, such as biopsies; biologicalfluids, such as cerebrospinal fluid; plasma; serum; urine; respiratorysecretions; bile synovial fluid; pleural fluid; lymph; tears; saliva; orstool samples. Such unpurified samples often contain amine contaminants,such as small amines, nucleophiles, or other unknown reactive compounds.For example, contaminants that are present during reductive amination insignificant amounts might create artifacts by competing with an agentsuch as 2-AB, thereby reducing the labeling of the glycans and theconsequent glycan signal during analysis. Similarly, the presence of thecontaminants during labeling of glycosylamines with an amine-reactivedye can result labeling of the contaminants, thereby creating labeledartifacts that interfere with the glycan signal of interest. It isexpected that adding mild electrophile prior to denaturation blocksthese contaminants in the sample, thereby reducing both of these typesof artifacts. In preferred embodiments, the denaturation includesheating the sample.

Optionally, the mixture is then subjected to a clean-up step, such aspassage through a solid phase cartridge, to remove the mildelectrophile. The labeled glycans are typically then provided to ananalytical means, such as an apparatus for performing high-pressureliquid chromatography, for capillary electrophoresis, for fluorescenceanalysis, or for mass spectrometry, to determine the types and amountsof glycans released from the glycoprotein or glycopeptide of interest orfrom those present in a particular biological sample. Often, a pluralityof samples are taken from the mixture so that the glycans released fromthe glycoprotein or glycopeptide of interest (or from a particularbiological sample) can be provided to, and analyzed by, a combination oftwo or more analytical means.

The scheme below sets forth a schematic showing the blocking of aminenucleophiles using DL-glyceraldehyde as an exemplar mild electrophile.

Use of Mild Electrophiles to Reduce Artifacts in Reductive Amination orin Non-Labeling Procedures

Glycans can also be labeled by reductive amination at their reducingends. Two labels typically used in the art for labeling by reductiveamination are 2-AA (anthranilic acid) and 2-AB (2-aminobenzamide). See,e.g., Bigge J.C., et al., Nonselective and efficient fluorescentlabeling of glycans using 2-aminobenzamide and anthranilic acid. Anal.Biochem., 230:229-238 (1995). Since this labeling method does not employamine-reactive dyes, it does not label the N-terminus of theglycoprotein or amine-containing side chains. However, the amount ofglycan available to be labeled by reductive amination can be reduced byinteractions of the glycans with reagents which may be present in thelabeling solution and which can react with the glycans. For example,Tris, or tris(hydroxymethyl)aminomethane, is commonly used as aconstituent of buffer solutions.

An excess of Tris in the buffer, however, can allow the Tris to competewith the label, such as 2-AB, in reacting with glycans present in thesolution. Similarly, if the solution containing the glycans has not beensubjected to sufficient clean-up steps, there could be small molecules,such as peptides, with an amine that may also be able to react with theglycans. Any reduction in the amount of glycans available to be labeledby reductive amination results in an understatement of the amount ofglycans present in the sample. It is believed that the presence of amild electrophile when the glycans are introduced into the solution(such as by chemical release from a glycoprotein) reduces theinteraction of the glycans with such reagents, thereby increasing theaccuracy of the analysis of the glycans released from the glycoproteinor glycopeptide. Typically, the mild electrophile is added to thesolution prior to denaturation of the glycoprotein or glycopeptide.

Since the presence of a mild electrophile can keep reagents or unknownsmall molecules in the solution from unwanted interactions with glycans,they can also be used to reduce artifacts in situations that do notinvolve a labeling reaction. For example, interaction of a glycan with asmall molecule in the solution could affect the apparent molecularweight of the glycan, throwing off subsequent analysis by massspectrometry (“MS”). Other analytical techniques known in the art thatdo not involve labeling the glycans but that can benefit by the presenceof a mild electrophile during denaturation of the glycoprotein orglycopeptide include: High-performance Anion Exchange Chromatographycoupled with Pulsed Amperometric Detection (“HPAE-PAD”), Charged AerosolDetection (“CAD”), and Evaporative Light Scattering Detection (“ELSD”).

Mild Electrophiles

In some preferred embodiments, the mild electrophile is an aldehyde,such as DL-glyceraldehyde, or glycoaldehyde dimer. In preferredembodiments, the mild electrophile prevents labeling of protein when aprotein is in a solution with an amine-reactive dye, and is compatiblewith the enzyme being used to deglycosylate the glycoprotein orglycopeptide. As used herein, “compatible with the enzyme being used”means that the mild electrophile does not denature the enzyme or reduceits activity to less than 80% of the activity the enzyme has in theabsence of the mild electrophile and more preferably does not reduce itsactivity to less than 90%, 91%, 92%, 93% 94%, 95%, 96%, 97% 98% or 99%of the activity of the enzyme in the absence of the mild electrophile,with each successive higher percentage of activity being more preferred.The percentage of activity of the enzyme of choice can be readilymeasured to test the compatibility of any particular mild electrophileby dividing a sample into a first and a second aliquot, adding the mildelectrophile being tested to the second aliquot but not to the firstaliquot, performing denaturation and labeling steps using anamine-reactive dye, and comparing the glycan labeling of the firstaliquot compared to that of the second aliquot. Mild electrophiles thatcause a reduction in the measurement of the glycans present in thesecond aliquot to that of the first aliquot are less preferred. Anexemplary assay is set forth in the Examples.

In a preferred embodiment, the mild electrophile is DL-glyceraldehyde,shown in Structure 1. As used herein, references to “glyceraldehyde”refer to DL-glyceraldehyde, unless otherwise specified or required bycontext.

In some embodiments, the mild electrophile is not L-glyceraldehyde. Insome embodiments, the mild electrophile is not D-glyceraldehyde.

In a second preferred embodiment, the mild electrophile isglyceraldehyde dimer (3,6-Dihydroxy-1,4-dioxane-2,5-dimethanol), shownbelow as Structure 2.

In a third preferred embodiment, the mild electrophile is glycoaldehydedimer, shown in structure 3. In some embodiments, the glycoaldehydedimer is DL-glycoaldehyde dimer.

As a reagent, glycoaldehyde dimer is sold as a crystalline powder andexists in the form of structure 2. According to Wikipedia, in aqueoussolution, glycoaldehyde dimer exists as a mixture of at least fourspecies, which rapidly interconvert.

In some embodiments, other aldehydes can be used as mild electrophilesin the inventive compositions, kits, and methods. These include:erythrose, pyruvaldehyde, 2-ethylbutyraldehyde, 2-methylbutyraldehyde,2-methylvaleraldehyde, 4-methyl-4-nitrovaleraldehyde, acetaldehyde,butyraldehyde, isobutyraldehyde, propionaldehyde, and valeraldehyde. Inpreferred embodiments, the aldehyde is not itself fluorescent. Anyparticular aldehyde or other mild electrophile can be tested, forexample in the assays taught in the Examples, to see if it preventsartifact peaks without interfering with the ability to detect labeledglycans.

Deglycosylating Glycoproteins and Glycopeptides

In the inventive methods, N-glycans are preferably released from theglycoprotein or glycopeptide of interest by enzymatic means. Protocolsand workflows for releasing N-glycans from glycoproteins usingdeglycosylation enzymes have been known in the art for years and it isassumed persons of skill are familiar with the ranges of times,temperatures and pH used in those workflows and protocols. An exemplarprotocol is set forth in the Examples.

Enzymatic digestion by enzymatic cleavage is typically achieved with anexoglycosidase, an endoglycosidase, or an amidase, such as PNGase F.Exemplar endoglycosidases include endo-alpha-N-acetyl-galactosaminidase,Endoglycosidase F1, Endoglycosidase F2, Endoglycosidase F3, andEndoglycosidase H. In some embodiments, the enzyme is the commondeglycosylation enzyme PNGase F(Peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase, EC 3.5.1.52),which releases N-glycans from the glycoprotein in the form ofglycosylamines. The glycosylamines can then be labeled with anamine-reactive dye, as discussed below. Glycosylamines hydrolyze overtime. Accordingly, N-glycans released from a glycoprotein asglycosylamines preferably are labeled within 30 minutes of beingreleased from the glycoprotein, with shorter periods, such as about 25minutes, about 20 minutes, about 15 minutes, or about 10 minutes, beingmore preferred, in that order, with “about” meaning ±2 minutes. Inpreferred embodiments, the N-glycans are labeled within about 9, 8, 7,6, 5, 4, 3, 2 or 1 minutes after being released from the glycoprotein,with “about” meaning ±30 seconds, with each successive shorter periodbeing more preferred. In particularly preferred embodiments, theN-glycans are labeled within 30 seconds after release from theglycoprotein.

Amine-Reactive Labels

As used herein, the terms “amine-reactive labels” and “amine-reactivedyes” are used interchangeably. Both are used to denote reagents that,under appropriate conditions, can covalently attach to an amine, such asthe side group of a lysine present on a protein or to a glycosylaminereleased from a glycoprotein by enzymatic digestion, to provide a tagwhich can then be detected by an analytical means of choice, such asHPLC or capillary electrophoresis.

A number of amine-reactive dyes are known in the art. The website ofThermo Fisher Scientific (Waltham, Mass.), lists 152 reagents under thelabel “Amine-Reactive Fluorophores, Biotins, Quantum Dots, & OtherLabels,” including 1-pyrenebutanoic acid, succinimidyl ester,2′,7′-Difluorofluorescein (Oregon Greene 488), 5(6)-CR 6G, SE(5-(and-6)-Carboxyrhodamine 6G, Succinimidyl Ester), mixed isomers, and7-Diethylaminocoumarin-3-Carboxylic Acid. ProZyme, Inc. (Hayward,Calif.) sells a variety of amine-reactive labels, including a basic dye,InstantPC™ (2-(diethylamino)ethyl4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate). Two acid labelsdisclosed in co-owned U.S. Pat. No. 8,124,792 are8-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,5-trisulfonicacid (amine-reactive “ANTS™”) and4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoic acid(“InstantAA™”). Amine-reactive ANTS™ and “InstantQ”(7-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,6-trisulfonicacid), another dye with three sulfonic acids, add 3 negative charges anda fluorophore to the glycans, which facilitates their separation bycapillary electrophoresis and detection. And, Waters Corporation(Milford, Mass.), sells RAPIFLUOR-MS® for labeling glycosylamines. (Thecompound sold as RAPIFLUOR-MS® is found in U.S. Published Patent Appin.US2014/0179011 A1, on page 14, top left column, in the embodiment whereR=N.) It is anticipated that mild electrophiles can be used effectivelywith each of these amine-reactive labels.

Conditions for Labeling and Analysis of Glycans Released asGlycosylamines

Persons of skill are familiar with methods for rapid labeling ofN-glycans released from glycoproteins as glycosylamines under mildconditions. U.S. Pat. No. 8,124,792 (the “'792 patent”), for example,discusses rapid labeling of N-glycans under mild conditions at col. 22,line 24, to col. 23, line 3, and col. 30, Example 10, to col. 32,Example 13, as do the corresponding portions of U.S. Pat. No. 8,445,292(each of these patents is incorporated herein by reference). Methods forproviding labeled glycosylamines to an analytical means are alsowell-known in the art, as exemplified by the teachings of the '792patent at col. 23, line 41 to line 67 and col. 32, Examples 13 and 14.Typically, the labeled glycosylamines are analyzed by an analyticalmeans such as high-pressure liquid chromatography, capillaryelectrophoresis, fluorescence analysis, mass spectrometry, or acombination of two or more of these means. In some embodiments, thecombination is of fluorescence analysis and mass spectrometry

Removal of the Mild Electrophile

In some embodiments, it may be desirable to remove the mild electrophilebefore subjecting the labeled glycans to analytical techniques such asmass spectrometry. A number of means for removal of reagents, such asmild electrophiles, are known in the art and can be used to remove themild electrophile prior to analysis of the labeled glycans. In preferredembodiments, the mild electrophile is removed by using a solid phasecartridge or other solid phase extraction (“SPE”) device used in the artto remove undesired components from a solution. The labeled glycans aretypically loaded onto a selected cleanup cartridge, washed to removemost non-glycan contaminants, and then eluted. Cleanup procedures forlabeled glycans are routinely used in the art and it is expected thatpractitioners are well familiar with them.

Kits

Conveniently, kits can be provided containing a selection ofdeglycosylation enzymes and one or more mild electrophiles, such asDL-glyceraldehyde or glycoaldehyde dimer, either in dried form or in asolution that can be added directly to a sample of a glycoprotein ofinterest prior to its denaturation. Conveniently, the kits can furthercomprise one or more amine-reactive dyes to be used in labelingglycosylamines released by enzymatic digestion of the glycoprotein, orreagents for conducting reductive amination of glycans, such as 2-AB, orboth an amine-reactive dye and a reagent, such as 2-AB, for performingreductive amination. The kits can further provide instructions fordenaturing the glycoprotein in the presence of the mild electrophile.

EXAMPLES Example 1

This Example sets forth abbreviations for some of the reagents used inexemplar workflows of deglycosylation and labeling procedures performedusing exemplar mild electrophiles in some of the Examples below.

“HEPES”: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid“TMD”: 1,3-Di(4-pyridyl)propane“DMF”: dimethylformamide

“ACN”: Acetonitrile “NMF”: N-methylformamide “TSH”: Bovine ThyrotropicHormone

“EPO”: EPOGEN® (epoetin alfa)

Example 2

This Example sets forth an exemplar workflow for denaturing anddeglycosylating a glycoprotein in the presence of an exemplar mildelectrophile, and labeling amines released from the glycoprotein byenzymatic digestion. Duplicate sets of assays were run, the first usingRNase b as the glycoprotein and the second using TSH as theglycoprotein.

Denaturation Step:

2 μl of 5% fatty acid detergent (tetramethyl ammonium laurate) was addedto 20 μl of a solution of 2 mg/ml protein (40 μg of protein).

Samples were divided into two aliquots. To the first aliquot, 2 μl of asolution of 100 mg/ml (DL)-glyceraldehyde in water was added. The secondaliquot did not have (DL)-glyceraldehyde added and served as thecontrol.

The reagents were thoroughly mixed and then heated to 90° C. for 3minutes to denature the glycoproteins. The sample was cooled to roomtemperature for 2 minutes.

Digestion Step:

A 2 μl aliquot of a 1:1 mixture of 1 mg/ml PNGase F:750 mM HEPES pH8 wasadded to each of the cooled samples and the samples were incubated at50° C. for 5 minutes to release the N-glycans. The samples were cooledto room temperature for 2 minutes.

Labeling Step:

A 1:1 mixture of 0.2 M 2-diethylaminoethyl4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate (“InstantPC™”).in DMF:1 M TMD in DMF was freshly prepared. Five μl of the mixture wasadded to the sample to label the released glycosylamines. The labelingwas allowed to proceed for 2 minutes.

Cleanup Step:

The labeled glycans were resuspended in 200 μl of acetonitrilecontaining 1% formic acid and the solution was loaded onto GlykoPrep®Cleanup Cartridges (ProZyme, Inc., Hayward,CA). The cartridges were spunat 300×g for 3 minutes. The cartridges were washed with 200 μl of a 2.5%solution of formic acid in ACN for an additional 3 minutes at 300×g. Thesamples were eluted with 100 μl of a solution of 160 mM ammoniumformate, pH 6.5 containing 10% ACN.

Analysis Steps

One μl of eluted sample was injected for high performance liquidchromatography (HPLC) analysis.

Example 3

This Example reports the results of the studies reported in Example 2.

FIG. 1 is a graph showing the results of a high performance liquidchromatography (“HPLC”) analysis of assays conducted using RNase b asthe exemplar glycoprotein and InstantPC™ as the dye. Emissions units, or“EU,” are shown on the Y axis, while time in minutes is shown on the Xaxis. As shown in the legend of the graph, the dashed line shows theresults in the absence of (DL)-glyceraldehyde in water (the “blocker”),while the solid line shows the results when the blocker was present.

FIG. 2 is a graph showing the results of a HPLC analysis of assaysconducted using Bovine Thyrotropic Hormone as the exemplar glycoproteinand InstantPC™ as the dye. As for FIG. 1, emissions units, or “EU,” areshown on the Y axis, while time in minutes is shown on the X axis. Asshown in the legend of the graph, the dashed line shows the results inthe absence of (DL)-glyceraldehyde in water (the “blocker”), while thesolid line shows the results when the blocker was present.

FIGS. 1 and 2 both show a dramatic reduction in artifacts on the rightside of the respective graphs in the assay run in the presence of theexemplar mild electrophile in comparison to the assay in the absence ofthe electrophile. As persons of skill will be aware, the glycans presenton RNase b and on bovine thyrotropic hormone are known. With respect toFIG. 1, the “hump” and peak of the dotted line to the right of the 6.60minute time point represent artifacts, not glycans. The hump is not seenin the solid line showing the presence of the blocker, and the peak atapproximately 9 minutes is far smaller. With respect to FIG. 2, thepeaks and long “hump” of the dotted line to the right of the 4.3 minutetime point represent artifacts, not glycans. The peaks betweenapproximately 4.3 minutes and 5.3 minutes seen in the absence of theblocker are much reduced or not present in the presence of the blocker,while the large hump between approximately 6.1 minutes and 8.6 minutesin the absence of the blocker is almost wholly absent in the presence ofthe blocker.

Example 4

This Example sets forth an exemplar workflow for denaturing anddeglycosylating an exemplar glycoprotein in the presence of an exemplarmild electrophile, labeling amines released from the glycoprotein with adifferent dye, and analyzing the labeled glycans (and any labeledprotein) using capillary electrophoresis.

Denaturation Step:

2 μl of 5% fatty acid detergent was added to 20 μl of a solution of 2mg/ml desalted RNase b or TSH (40 μg of protein). For half of thesamples, 2 μl of a solution of 100 mg/ml (DL)-glyceraldehyde (“theblocker”) in water was added to the mixture; as a control, no blockerwas added to the other half of the samples. The reagents were thoroughlymixed and then heated to 90° C. for 3 minutes to denature theglycoproteins. The samples were then cooled to room temperature for 2minutes.

Digestion Step:

A 2 μl aliquot of a 1:1 mixture of 1 mg/ml PNGase F:750 mM HEPES pH8 wasadded to each of the cooled samples and the samples were incubated at50° C. for 5 minutes to release the N-glycans. The samples were cooledto room temperature for 2 minutes.

Labeling Step:

A 1:1:2 mixture of 0.05 M InstantQ™ in DMF:1 M TMD in DMF:NMF wasfreshly prepared. Twenty μl of the mixture was added to the samples tolabel the released glycosylamines. The labeling was allowed to proceedfor 2 minutes.

Cleanup Step:

A cleanup plate was prepared by rinsing the plate with 400 μl 5% Formicacid, 95% water. The cleanup plate was rinsed with 3× 400 μl 95%Ethanol, 5% Formic acid. 400 μl of 100% ethanol was loaded to thecleanup plate, without vacuum. 150 μl of 100% ethanol was added to thesamples, which were mixed several times and loaded onto the top of the400 μl ethanol in the cleanup plate and immediately mixed. Vacuum wasapplied to load and the plate was washed 2× with 400 μl Ethanol, andeluted with 150 μl water prior to analysis using capillaryelectrophoresis.

Example 5

This Example reports the results of the studies reported in Example 4.

FIG. 3 shows an analysis by capillary electrophoresis of the glycansreleased from RNase b in the presence (solid line) or absence (dashedline) of the blocker. The two lines have been offset on the Y-axis tofacilitate the ability to see each line. Artifacts, presumably fromlabeling of the agycosylated protein, are much reduced.

Example 6

This Example reports the effect of studies of the effect of an exemplarmild electrophile on assays using a different labeling reagent and adifferent exemplar glycoprotein.

Denaturation and Deglycosylation Steps

RapiGest™ SF (Waters Corp., Milford, Mass.), 5% (w/v) was prepared bydissolving 10 mg of RapiGest™ SF Surfactant in 200 μl of Rapid Buffer ina tube and vortexing. 15.3 μl of water was dispensed into a 1 mL tube.7.5 μl of 2 mg/mL EPO protein was added into the tube containing theRapiGest™ SF. Six μl of buffered, 5% (w/v) RapiGest™ SF solution wasadded to the tube, and aspirated to mix. For half of the samples, 2 μlof a solution of 100 mg/ml (DL)-glyceraldehyde (“the blocker”) in waterwas added to the mixture, while the other half of the samples did nothave blocker added. The samples were heated at least to 90° C. for 3minutes and then cooled at room temperature for 3 minutes. 1.2 μl ofRapid PNGase F was added to the samples and aspirated to mix. Thesamples were incubated at 50° C. for 5 minutes and then cooled at roomtemperature for 3 minutes.

Labeling of Glycosylamines Step

335 μl of anhydrous DMF was added directly to one vial of 23 mg ofRAPIFLUOR-MS® label (Waters Corp., Milford, Mass.) and mixed tosolubilize. Twelve μl of the reagent solution was added to thedeglycosylation mixture of the previous step and aspirated to mix andallow labeling. The labeling was allowed to proceed at room temperaturefor 5 minutes. The solution was then diluted with 358 μl of acetonitrile(ACN) and aspirated to mix.

Clean-Up Step

A GLYCOWORKS® HILIC pElution Plate (Waters Corp.) was set up, and shimsor spacer and waste tray added. Wells were conditioned by adding 200 μlof water per well. Wells were equilibrated by adding 200 μl 85% ACN.ACN-diluted samples were loaded (˜400 μl per well). Wells were washedwith two 600 μl volumes of 1% formic acid, 90% ACN. The waste tray wasreplaced with a sample collection tray loaded with 600 μl tubes. Glycanswere eluted with three 30 μl volumes of Water’ solid phase elution(“SPE”) buffer into 600 μl tubes. SPE eluate was diluted with 100 μl ofDMF and 210 μl of ACN and aspirated to mix prior to HPLC analysis.

Example 7

This Example discusses the results of the studies reported in Example 6.

FIG. 4 shows the HPLC analysis of the samples discussed in Example 6.The vertical arrows show three points at which the presence of theblocker eliminates or sharply reduced artifacts compared to the sameassay in the absence of the blocker.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-41. (canceled)
 42. A kit for labeling glycosylamines released from aglycoprotein or glycopeptide, said kit comprising, (a) a mildelectrophile, (b) an amine-reactive dye, an agent for performingreductive amination, or both an amine-reactive dye and an agent forperforming reductive amination, and (c) instructions on denaturing saidglycoprotein or glycopeptide in the presence of said mild electrophile.43. The kit of claim 42, wherein said agent for performing reductiveamination is anthranilic acid (“2-AA”) or 2-aminobenzamide (“2-AB”). 44.The kit of claim 42, further comprising a deglycosylation enzyme. 45.The kit of claim 44, further wherein said deglycosylation enzyme isPNGase F.
 46. The kit of claim 42, wherein said mild electrophile isDL-glyceraldehyde, glyceraldehyde dimer, glycoaldehyde dimer, erythrose,pyruvaldehyde, 2-ethylbutyraldehyde, 2-methylbutyraldehyde,2-methylvaleraldehyde, 4-methyl-4-nitrovaleraldehyde, acetaldehyde,butyraldehyde, isobutyraldehyde, propionaldehyde, or valeraldehyde. 47.The kit of claim 42, wherein said mild electrophile isDL-glyceraldehyde, glyceraldehyde dimer, or glycoaldehyde dimer.
 48. Thekit of claim 47, wherein said mild electrophile is DL-glyceraldehyde.49. The kit of claim 42, wherein said amine-reactive dye is selectedfrom the group consisting of (2-(diethylamino)ethyl4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoate) (InstantPC™),8-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,5-trisulfonicacid (“InstantANTS™”),4-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]benzoic acid(“InstantAA™”),(7-[(2,5-dioxopyrrolidin-1-yl)oxycarbonylamino]naphthalene-1,3,6-trisulfonicacid) (“7-ANTS”), and RAPIFLUOR-MS®.